Cell cycle and apoptosis were analyzed using flow cytometry as previously described by our group [13 (link)]. The cell-cycle analysis kit and Annexin V-PE/7-AAD apoptosis kit were obtained from Keygen Biotech (Nanjing, China). Operations were carried out according to the kit instructions.
Cell cycle analysis kit
Manufactured by Keygen Biotech
Sourced in China
The Cell Cycle Analysis Kit is a laboratory equipment designed for the analysis of cell cycle progression. It provides the necessary tools and reagents to quantify the distribution of cells in different phases of the cell cycle, such as G1, S, and G2/M. The kit includes a set of dyes, buffers, and protocols to prepare and analyze cell samples using flow cytometry or other compatible techniques.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Annexin 5 pe 7 aad apoptosis kit, by Keygen Biotech (1 mentions)
Fasc calibur, by BD (1 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Erlotinib hcl osi 744, by Selleck Chemicals (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Transgene (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Flow cytometry, by Beckman Coulter (1 mentions)
Accuri c6, by BD (1 mentions)
Transwell chamber, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Matrigel, by Corning (1 mentions)
Ecl plus kit, by GE Healthcare (1 mentions)
Sybr green pcr master mix, by Takara Bio (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Annexin 5 fitc pi, by BD (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Keygen Biotech (1 mentions)
12 protocols using cell cycle analysis kit
1
Cell Cycle and Apoptosis Analysis
2
Apoptosis and Cell Cycle Analysis
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Apoptosis assays were performed using the FITC Annexin V Apoptosis Detection Kit I (BD, USA) following the recommended procedure. Cells were harvested 48 h after blue laser irradiation at 4 J/cm2, 8 J/cm2, or no irradiation and washed twice with PBS. Subsequently, cells were resuspended in 1X binding buffer and stained with Annexin-V-FITC and PI for 15min. Samples were analyzed by flow cytometry (Beckmancoulter, USA). The apoptosis cells were considered as the upper right quadrants.
Cell cycle assays were performed using the Cell Cycle Analysis Kit (KGA512, KeyGEN BioTECH, China) following the recommended procedure. Cells were harvested 48 h after blue laser irradiation at 4 J/cm2, 8 J/cm2, or no irradiation and fixed with 70% ethanol at 4°C overnight. Next, each sample was stained with working solution containing 450 μl propidium iodide and 50 μl RNase A for 30 min. A flow cytometer (FASC Calibur, BD, USA) was used to detect cell cycle distribution, and 15,000 events were measured for each sample.
Cell cycle assays were performed using the Cell Cycle Analysis Kit (KGA512, KeyGEN BioTECH, China) following the recommended procedure. Cells were harvested 48 h after blue laser irradiation at 4 J/cm2, 8 J/cm2, or no irradiation and fixed with 70% ethanol at 4°C overnight. Next, each sample was stained with working solution containing 450 μl propidium iodide and 50 μl RNase A for 30 min. A flow cytometer (FASC Calibur, BD, USA) was used to detect cell cycle distribution, and 15,000 events were measured for each sample.
3
Apoptosis and Cell Cycle Analysis
4
Cell Cycle Analysis by Flow Cytometry
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Cell cycle assays were performed using a cell cycle analysis kit (KeyGEN Biotech, Nanjing, China) in accordance with the manufacture's instructions. Cells were seeded at 105 cells per well into six-well plates. After 72-h treatment with 4-OHT, cells were harvested and rinsed with ice-cold phosphate buffered saline (PBS) and fixed in 70% ethanol at 4 °C overnight. After PBS washes, the cells were incubated with RNase A for 30 min at 37 °C, stained with propidium iodide (PI), and analyzed by flow cytometry (Beckman Coulter, Inc., Brea, CA, USA). The cell cycle distribution was assessed using Modifit software.
5
Cell cycle analysis of TEM8 knockdown
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U2OS and MG63 cells (3 × 105) were seeded in each well of six-well plates and incubated overnight until 30–50% confluent, then transfected withTEM8 siRNA or scrambled siRNA for 6 h. The cells were harvested for test at 24–48 h after transfection. For stably transfected with shRNA1 and shNC, cells were incubated to reach 90% confluent until harvested. Then cells were washed with cold PBS, fixed with 70% ethanol for overnight at 4 °C, and conducted using cell cycle analysis kit(Keygen, Nangjing, China). Finally, cells were analyzed by a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ) with Cell Quest Pro software. This experiment had three independent replicates.
6
Cell Apoptosis and Cell Cycle Analysis
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Cell apoptosis was examined using Annexin V-PE and 7-AAD staining assays (BD Bioscience, USA) following the manufacturer’s protocols, and cell cycle was analyzed using a Cell Cycle Analysis Kit (Keygen Biotech, China). Cell cycle and apoptosis were detected by flow cytometry (FCM) analysis (Accuri C6, BD Bioscience, USA).
7
Characterization of NSCLC Cell Line H1299
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The non-small cell lung cancer (NSCLC) cell line H1299 was cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum. All overexpression plasmids were constructed by Chengdu Yeda Technology Co., Ltd. A transfection kit (jet PRIME, 21Y0910L3) was used to transfect plasmids into cells for 48 h. Cell proliferation was detected with a Cell Counting Kit (CCK8; Zeta, Z0207205508C, Shanghai, China). Cells were seeded into Transwell chambers (8 μm, Millipore Corporation, USA) with (invasion) and without (migration) 10 mg/mL Matrigel (354,248, Corning, Jiangsu, China) precoated in 10% fetal bovine serum RPMI-1640 medium. Cells that migrated or invaded through the membrane and attached to the bottom of the Transwell plates were counted 48 h post seeding. A Cell Cycle Analysis Kit (KeyGEN BioTECH, KGA512, Jiangsu, China) and Cell Apoptosis Kit (KeyGEN BioTECH, KGA108) were used to assess the cell cycle distribution and apoptosis, respectively.
8
Evaluation of MTT Cytotoxicity Assay
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3-(4 (link),5 (link))-dimethylthiahiazo(-z-yl)-3,5-di-phenytetrazoliumromide (MTT) was purchased from Shanghai Dingguo Biological Technology Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). TRIzol® Reagent and Lipofectamine® 2000 were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). M-MLV Reverse Transcriptase was purchased from Promega Corporation (Madison, WI, USA) and SYBR®-Green PCR Master mix was purchased from Takara Bio Inc. (Shiga, Japan). A Cell cycle analysis kit and an apoptosis kit were purchased from Nanjing KeyGen Biotech., Co., Ltd. (Nanjing, China). An ECL-PLUS™ kit was purchased from GE Healthcare (Piscataway, NJ, USA).
9
Apoptosis and Cell Cycle Analysis
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A375 (2.5 × 105 cells per well) cells were seeded in 6-well plates with 0, 2, 8 and 16 μM Cu6NC for 24 h. The cells were collected using trypsin–EDTA and stained with annexin V-FITC/PI (BD Biosciences, USA) for 15 min, and then apoptotic cells were identified by flow cytometry. The cell cycle was measured by PI staining using a cell cycle analysis kit (KeyGen Biotech, China). After staining following the manufacturer’s instructions, the cells were analyzed by flow cytometry (FACS Aria III, USA). Data were processed by ModFit LT software.
10
Cell Cycle and Apoptosis Analysis
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Cell cycle was detected by flow cytometry using a cell cycle analysis kit (KeyGEN, Nanjing, China) according to the protocol. The proliferative index was calculated by the formula for (S + G2M)/(G0G1 + S + G2M).
Cell apoptosis was detected by flow cytometry using an Annexin V fluorescein isothiocyanate kit (APC/7-AAD) (KeyGEN, Nanjing, China) according to the protocol.
Cell apoptosis was detected by flow cytometry using an Annexin V fluorescein isothiocyanate kit (APC/7-AAD) (KeyGEN, Nanjing, China) according to the protocol.
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